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. 2017 Oct 19;7:13605. doi: 10.1038/s41598-017-13997-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Quantitative, qualitative, and functional analyses of lifetime Nkcc1 knockdown effects in Actin-tTS::Nkcc1 ^tetO/tetO mice (WL-KD mice). (a) At P35, cochlear Nkcc1 transcription is decreased in WL-KD mice vs. controls in comparison with other K⁺ circulation molecules as labeled. mRNA levels are normalized to Gapdh. Nkcc1 mRNA was significantly suppressed in WL-KD mice compared with controls, but the mRNAs of other K⁺ circulation molecules were not suppressed. (b–e) In situ hybridization of Nkcc1 mRNA in mouse cochleae. (b) Normal Nkcc1 mRNA expression in cochlea of control. Arrows show normal SV thickness. (c) Magnification of view in (b). (d) Strong suppression of Nkcc1 level in WL-KD mice. (e) Magnification of view in (d). (f) Nkcc1 knockdown significantly elevates the ABR threshold, which indicates hearing loss. **Significant difference at p < 0.01 (Mann-Whitney test). NS: no significant difference (Mann-Whitney test).